Updated FTP location. Small extracellular vesicles (sEVs) are important mediators of intercellular communication with respect to diverse pathophysiological processes. The heterogeneity of sEV populations is a key factor in pursuing the sEV-related translational and basic research into forward but remains challenging for unraveling. Here, we discovered novel phosphatidylserine (PS; marker for sEV uptake by macrophage)-deficient sEV subpopulations which possessed super long blood circulation through escape from the uptake by macrophages (main player of the rapid sEV clearance from blood). PS(-)-sEVs were identified in various cultured-cells as a minor population. Meanwhile, circulating somatic cell-derived sEVs in the blood were found to be majorly PS(-)-sEVs, which were caused by rapid uptake of PS(+)-sEVs by macrophages within 10 min after entry into the circulation. These results suggest endogenous PS(-)-sEVs could truly be the key player of sEV-mediated intracellular communication and that PS(-)-sEVs can be a good target for sEV-based diagnosis as well as a potent candidate for sEV-based delivery carrier. Our findings shift the paradigm for further understanding the biology as well as for the translational applications of sEV.